Systems, devices, and methods for automated sample thawing

ABSTRACT

The present invention generally relates to thawing a cryogenically frozen sample. A method of thawing a sample includes receiving a temperature data feed from one or more temperature sensors reporting an exterior surface temperature of a vessel, calculating a thaw end time based on the temperature data feed, and outputting a signal to interrupt thawing of the sample at the calculated thaw end time, the sample at the thaw end time having solid phase remaining. A method of thawing a cryogenic sample includes heating a sample container with a heater, determining a thaw start time for the cryogenic sample; determining an estimated thaw end time of the cryogenic sample based on the determined thaw start time of the cryogenic sample, the cryogenic sample at the estimated thaw end time having solid phase remaining; and stopping the heating of the sample container after the estimated thaw end time.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 15/602,711, filed May 23, 2017, which claims priority to U.S.Pat. No. 10,555,374, filed May 14, 2015, which claims priority to U.S.Provisional Application No. 61/994,586 filed May 16, 2014, and U.S.Provisional Application No. 62/042,669 filed Aug. 27, 2014, the fulldisclosures which are incorporated herein by reference in their entiretyfor all purposes.

BACKGROUND OF THE INVENTION

The present invention generally relates to the cryogenic preservation ofceils and more specifically to systems, devices, and methods for therecovery of cryogenically-preserved cells and tissue

Cryogenic preservation of ceils in suspension is a well-established andaccepted technique for long term archival storage and recovery of livecells. As a general method, cells are suspended in a cryopreservationmedia typically including salt solutions, buffers, nutrients, growthfactors, proteins, and cryopreservative. The cells are then distributedto archival storage containers of the desired size and volume, and thecontainers are then reduced in temperature until the container contentsare frozen. Typical long-term archival conditions include liquidnitrogen vapor storage where temperatures are typically between −196 and−150 degrees Celsius.

The successful recovery of live cells preserved by such methods may bedependent upon minimizing injurious ice crystal growth in theintracellular region during both the freezing and thawing processes Someadvances have been made to reduce intracellular ice crystal growthduring the freezing process. For example, intracellular ice crystalgrowth may be reduced by adding a cryoprotectant compound to the tissuesor cell suspension solution that inhibits ice crystal nucleation andgrowth both extracellularly and intracellularly. Additionally, thegrowth of intracellular ice can be controlled through management of therate of sample temperature reduction. During the freezing processextracellular ice crystal formation will exclude solutes and cells fromthe developing ice crystal structure thereby concentrating the solutesand cells in the remaining liquid phase. The increase in soluteconcentration will establish an osmotic potential that will promote thedehydration of the cells while allowing time for cell membrane-permeablecryoprotectants to equilibrate in concentration within the intracellularvolume. As the freezing process progresses a temperature will be reachedat which the high solute concentration will solidify in a glass statewith minimal size of ice crystal nuclei within the intracellular volume.The solid-state cell suspension is then further reduced in temperatureuntil the cryogenic storage temperature is reached. At this temperaturemolecular activity is sufficiently reduced that the cells may be storedindefinitely. For optimal cell recovery following cryogenic storage, therate of temperature reduction during the freezing process must fallwithin a range of values. If the temperature reduction rate is too fast,the cells may freeze before the level of intracellular water has beensufficiently reduced, thereby promoting the growth of intracellular icecrystals. If the rate of temperature reduction is too slow, the cellsmay become excessively dehydrated and the extracellular soluteconcentration may become too high, with both cases leading to damage ofcritical cellular structures. For this reason, the temperature reductionrate during the freezing process is typically controlled. For example,one method of controlling the rate of temperature reduction includessurrounding the sample with an insulating material and placing theassembly in a static temperature environment, while another methodincludes placing the exposed sample container into an isolation chamberin which the interior temperature is reduced at a controlled rate.

Returning the sample from the cryogenic archival state involves thawingthe sample to a fully liquid state. During the thawing process, againthe rate of temperature change can influence the viability of thecryogenically preserved cells. The solid contents of the sample storagevessels contains large islands of crystallized water which areinterposed by channels of glass state aqueous solutes intermixed withsmall nuclei of ice crystals. During the transition from the cryogenicstorage temperature to the conclusion of the phase change to acompletely liquid state, there is an opportunity for rearrangement ofthe water molecules within the sample including a thermodynamicallyfavored extension of the small ice nuclei within the cells. As thegrowth of the intracellular ice crystals have an associated potentialfor cell damage, and as the degree of crystal growth is a time-dependentthe phenomenon, minimizing the time interval of the transition throughthe phase change is desirable. A rapid slew rate in the sample vesseltemperature is typically achieved by partial submersion of the vessel ina water bath set to a temperature of approximately 37 degrees Celsius.Although a faster rate of thawing can be achieved by increasing thetemperature of the bath, submersion of the vessel in the bath willestablish temperature gradients within the vessel with the highesttemperatures being located at the vessel wall. As a result, transientthermodynamic states will occur wherein the temperature of theliquid-solid mixture will exceed the melting temperature even thoughfrozen material is present in close proximity. The intra-vesseltemperature gradient therefore places an upper limit on the bathtemperature. In addition, as common cryoprotectants have a known toxicinfluence on the cells, differential exposure of the cells in the liquidstate with respect to time and temperature allows for variation in theviability of the cells upon completion of the thaw process. As the toxiceffect of the cryoprotectants is enhanced at elevated temperatures, alower liquid temperature is desirable. For this reason, common thawingprotocols typically include a rapid thaw phase that is terminated when asmall amount of solid material still remains in the sample container.Following removal from the water bath, the sample temperature willquickly equilibrate to a temperature that is near to the phase changetemperature. Thawing protocols typically seek to minimize the durationat which the thawed sample is held in a state where the cryoprotectantis concentrated, and subsequent steps to dilute the sample or exchangethe cryopreservation media for culture media are commonly applied in asshort of an interval as possible. As the current methods and solutionsfor thawing of cryogenic samples in sample vials is dependent upon themethodology, protocols, and equipment that differs on an individualbasis, no current method is available by which the vial thawing processcan be standardized across the academic or clinical community.Accordingly, improvements may be desired.

SUMMARY OF THE INVENTION

The terms “invention,” “the invention,” “this invention” and “thepresent invention” used in this patent are intended to refer broadly toall of the subject matter of this patent and the patent claims below.Statements containing these terms should be understood not to limit thesubject matter described herein or to limit the meaning or scope of thepatent claims below. Embodiments of the invention covered by this patentare defined by the claims below, not this summary. This summary is ahigh-level overview of various aspects of the invention and introducessome of the concepts that are further described in the DetailedDescription section below. This summary is not intended to identify keyor essential features of the claimed subject matter, nor is it intendedto be used in isolation to determine the scope of the claimed subjectmatter. The subject matter should be understood by reference toappropriate portions of the entire specification of this patent, any orall drawings and each claim.

For thawing cells, conventional practice is to warm the cells quickly ina warm water bath (e.g., 37 deg. C.) to just about the point at whichthe last bit of ice is about to melt and then to dilute the cells slowlyinto growth media. If the sample is allowed to get too warm, the cellsmay start to metabolize, and be poisoned by the DMSO (dimethylsulfoxide) that is used in the freezing process. Generally, the thawingof cryogenically preserved cells and tissue is performed by labtechnicians and the applied protocol can not only vary between each labtechnician, but may also be technique dependent. The completion ofsample thaw is generally subjectively judged by each individualtechnician and may result in variation in the thaw rate or samples whichhave been allowed to become too warm. Although a repeatable thawingprofile is theoretically possible to achieve using a bath and manualcontrol of the vial insertion, expected variance in both technique anddegree of protocol compliance, particularly combined with therequirement to frequently remove the vial from bath to monitor the thawstatus, makes deviation from the standard profile a near certainty. Theremoval of the vial from the bath interrupts the thermal energy transferfrom the bath water to the vial and visual assessment of the thaw statusis often difficult and may be complicated by the presence of vial labelsand printed writing surfaces that are provided as integrated features ofthe vial product. Further water baths are also a source of contaminationand inadvertent submersion of the vial body-cap junction can result inthe introduction of bath liquid into the vial contents during removal ofthe vial cap.

Systems, devices, and methods that provide simplified, automated, and/ormore consistent sample thawing may be advantageous and may increase cellrecovery. Furthermore, a device that functions autonomously incombination with a methodology that is easily configured and performedmay provide a manner by which a standard thawing process may beintegrated across academic and/or clinical communities, therebyeliminating a source of variance in experimental results and therapeuticoutcome. Devices and methods that will provide automation andstandardization of the thawing process for sample vials may resolvemultiple obstacles in the execution of this achievement. The embodimentsof the present invention may address one or more of these issues. Toreplicate a thawing process in which the warming phase is terminatedafter substantial thawing (e.g., while a small portion of the sample isstill in the solid phase or when the sample substantially completes thephase change from solid to liquid), a thawing system may use one or moresensors and/or thawing algorithms to predict various stages of thethawing process. In some embodiments, as there is a range ofsusceptibility to the combined effects of intracellular ice crystalgrowth during thawing, cryoprotectant exposure, and liquid statetemperature elevation during the thaw process, obtaining a consistentrecovery state for a given cell type or cell mixture may be dependentupon close control of the thawing temperature increase profile withrespect to time.

Accordingly, in some embodiments of the invention, systems and methodsmay be provided for thawing samples under consistent and uniformconditions. As samples stored under cryogenic temperatures will often bein a location that is remote from the heater in which recoveryoperations will be conducted, a system must be established to regulatethe temperature of the sample during transport to ensure that the sampledoes not begin thawing prematurely or spend an unnecessarily longinterval at temperatures above −75° C. Although ideally the temperatureof a sample in the time interval between the retrieval from cryogenicstorage and the start of the thawing process should be maintained belowthe glass transition temperature of the cryogenic storage fluid(approximately −150° C.), however for a number of cell cultures, atemporary storage interval in the temperature range of −150° C. to −75°C. for at least several days may be utilized without a detectabledecrease in cell viability upon recovery and culture. For such samples,a transportation and temporary holding temperature of approximately −75°C. is readily applied as the temperature coincides with the phase changetemperature of solid carbon dioxide which may be used as a refrigerant.For more temperature-sensitive samples, a transportation and temporarystorage temperature of approximately −195° C. may be obtained by usingliquid nitrogen as a refrigerant. A system may include a container for asample vial that will allow thermal equilibration of the sample at atemperature at or below −75° C. The system may also include a containerfor the sample vessel for holding and thawing a sample. In someembodiments, a contact surface of the sample holder (hereafter referredto as a “warming block”) that is in physical contact with the outersurface of the sample vessel may be heated to a constant temperature(e.g., 37° C.). A micro-processor may be coupled with the samplecontainer holder and the processor may use a predictive thawing model toidentify an end time of the sample thawing process. The predictivethawing model may identify a thaw completion time based in part on thestarting time of the thawing process. In some embodiments, the thawcompletion time may be obtained from a pre-determined average thaw timefor a particular sample container format containing a particular samplevolume payload for a particular warming temperature, as by use ofreference to a look-up data table derived from experimental values. Inother embodiments the instant invention may receive data from atemperature sensor that rests in contact with the exterior surface ofthe sample vessel, and may determine the beginning of the sample solidto liquid phase conversion based on the temperature data, and incombination with a phase conversion interval that is experimentallyderived for an equivalent sample mass and vial configuration, predictthe time at which the phase change conversion will be completed or benear to completion. In other embodiments, the thaw completion time maybe determined entirely through a predictive calculation based uponanalysis of data received from a temperature sensor that is operablycoupled with the sample vessel (e.g., in direct contact with theexterior surface, through use of a non-contact infrared sensor, or thelike). In other embodiments, the approach of the end of the phase changeinterval will be detected by a noise signal in the data stream derivedfrom random motion of the solid phase remnant in the vial as detected bya temperature sensor that is operably coupled with the sample vessel. Insome embodiments the vial exterior surface temperature sensor is incontact with the side of the vessel while in other embodiments, thesensor is in contact with the bottom surface of the vessel. In otherembodiments, the temperature of the sample vessel contents will bemeasured by a sensor that is physically centered in the interior of thevessel and is isolated from the contents by a covering of material thatis a continuous extension of the vessel exterior. In some embodiments,the external vial surface sensor may be a component of the thawingdevice, while in other embodiments the sensor may be a component of thevessel. When the sensor is a component of the sample vessel, the sensormay be include a connection (e.g., electrical, radio, or opticconnection) to the thawing device by which data may be exchanged. Insome embodiments, the data stream may comprise thermometric dataexclusively while in other embodiments, the data stream may compriseadditional information such as, but not limited to, vessel trackinginformation, vial contents history and composition, and chain of custodyhistory. In some embodiments the temperature sensors may bethermocouples, thermistors, and resistance sensors, while in otherembodiments, the vial temperature may be detected by an infra-rednon-contact temperature detector.

Some embodiments may comprise one or more temperature transducers bywhich the temperature of the warming block may be monitored. In someembodiments the temperature of the warming block is controlled by amicroprocessor receiving temperature signal feedback from the warmingblock temperature transducers. The system may include one or moretransducers for recording one or more temperatures of the sample and/orthe sample container. In some embodiments one or more transducers may beprovided for measuring and/or recording the temperature of the exteriorsurface of the sample container. In some embodiments, one or moretransducers may be provided for measuring and/or recording thetemperature of the sample.

In some embodiments, the system may have a user interface and mayreceive a user input via the user interface before recordingtemperatures from the sample and/or sample container. Alternatively, thesystem may be automatically triggered to begin recording temperaturesfrom the sample and/or sample container after the sample container isinserted into the sample holder. In some embodiments, the system may usethe automatic triggering mechanism alone to signal the beginning of thethaw interval. In some embodiments, the system may use the automatictriggering mechanism to signal the beginning of the thaw interval, andin combination with the starting signal use a time value constant todetermine the completion of the thaw process. In other embodiments, analgorithmic analysis of temperature measurements of the outer surface ofthe sample container is used to determining the start and end-point ofthe interval of the sample solid to liquid phase change. The temperatureincrease during the interval where the sample is exclusively in a solidphase may be modeled by a linear time-invariant lumped system equationwherein a time constant value controls the rate of temperature increase.In other embodiments, the start of the thaw interval may be determinedby calculation of the time constant variable for a linear time-invariantlumped system equation such that the temperature output values of theequation will overlay the solid phase portion of the time-temperaturedata received from the vial surface temperature sensor, and by using amultiple of the time constant value to indicate the time of commencementof the solid to liquid phase change. In other embodiments, the automatedthawing system may use a combination of thaw insertion signal, a timevalue, and an algorithmic analysis of the sample container surfacetemperature data to determine the beginning and the end-point of thethaw process.

In some embodiments, following retrieval from the cryogenic storagesystem, the temperature of the frozen sample vial is allowed toequilibrate to a temperature of approximately −78° C. to −75° C. byplacing the sample into an aluminum alloy holder (the “equilibrationblock”) that is resting on and surrounded by dry ice. In thisembodiment, the vial is removed from the equilibration block andimmediately placed into the warm block of known and constanttemperature. Using this embodiment and method, a very uniform andpredictable thaw interval (the “total thaw interval”) can be determinedfor a given vial geometry and payload, thereby allowing a thawingcompletion to be predicted exclusively by a time interval followinginsertion into the warming block. In other embodiments, followingtemperature equilibration in the equilibration block and insertion intothe warming block, the thawing interval may be determined by acombination of total thaw time interval and algorithmic analysis of thevial exterior temperature data, thereby providing an internalself-reference check for the thaw interval prediction system.

In some embodiments, the warming block is configured to accept multiplesample container geometries (e.g., vials, vessels, bags, or the like) byproviding multiple receiver wells of the appropriate dimensions. Inother embodiments, the warming block is configured to accept only onesample container and is dedicated to a thawing sample container of theappropriate dimensions for that warming block only. In other embodimentsthe warming block is designed to accept multiple sample container sizesand geometries by the exchange of appropriate vial adapters designed forthe specific sample containers. Optionally, the warming block mayutilize morphing contacts, flexible wraps, rotating block faces, heatrollers, IR heaters, and/or scroll jaw chucks for accepting multiplesample container sizes and geometries. In some embodiments the systemmay adjust a completion time based in-part on a sample container type.Optionally, the system may be configured to automatically determine thetype of sample container being thawed. For example, some samplecontainers may include 1D barcodes, 2D barcodes, RFID chips, or othercomputer readable indicia that are readable by a barcode reader of thesystem or a RFID chip sensor of the system. The container's barcode,RFID chip, or other computer readable indicia may be linked to a thawingprofile of the container and the system may automatically determine athawing interval specific for the container or otherwise determine athaw end time specific for the type of container (e.g., through look uptables, formulas, or the like). In some embodiments, a look-up table maybe defined by a mathematical function. Optionally, the system mayreceive user input via a user interface for defining the type of samplecontainer being thawed. In some embodiments, the thaw end time and totalthaw time may be determined using the starting vessel externaltemperature, the temperature of the warming blocks (e.g., non-pliablesolid material), and the vessel type.

In some embodiments, the sample vial-receiving well of the warming blockcomprises a thermally conductive pliable material lining to provide auniform and repeatable level of thermal contact with the samplecontainer. In some embodiments the warming block is divided into twoparts such that when the two parts are separated, the interior walls ofthe sample container receiving well do not contact or have minimalcontact with the sample container, thereby facilitating the insertionand removal of the sample container from the well, and providing amanner with which to initiate the thawing process at a defined moment,in addition to providing a way of interrupting the flow of thermalenergy from the warming block to the sample vial. In some embodiments,the sample vial-receiving well of the warming block is divided by avertical plane that is coincident with the central axis of the samplereceiving well. In other embodiments the warming block is divided intomore than two parts (e.g., three parts, four parts, or more) that allowa space to be selectively introduced between the warming block samplevial receiver well wall and sample vial exterior by transient lateral orangular displacement of the warming block segments. In some embodiments,the warming block segments are constrained by mechanical linkages suchas slide mechanisms, hinged joints, kinematic linkages, hydraulicmechanisms, electrical solenoid mechanisms, screw mechanisms, magneticlinkages, or any combination thereof. In some embodiments the separatedparts of the warming block are automatically closed upon insertion ofthe sample vial into the warming block sample vial receiver well toefficiently contact the sample container on all or most of the lateralsurfaces of the sample container.

In some embodiments the invention will provide audio or visual feedbackto allow the user to be informed of the device state and the status ofthe sample thawing process. In some embodiments, upon reaching thedesired end-point of the melting process, the invention will alert theuser using a visual and audio signal.

In some embodiments, the invention will be dedicated to a general typeand shape of sample vial with no available user input other thanselection of an on-off state. In other embodiments, the invention willaccept input from the user. In some embodiments, the predictive thawingmodel may, without limitation, be adjusted by user input to account forthe type of the sample container holding the sample, presence of a labelon the sample container, a sample container fill level, and/or age ofthe thermally conductive medium between the warming block and thevessel.

Optionally, the system may provide an alert to a user when the meltingsample reaches a desired level of remaining solid phase. In someembodiments, the desired end-point is a state wherein solid phase is asmall fraction of the starting amount of solid material. In otherembodiments, the desired end-point of the thaw process may be when thesolution is completely liquid. In some embodiments, the end-point alertprovided by the system may be an audio or visual indicator orcombination of audio and visual signals. In other embodiments, anend-point alert may be wirelessly transmitted to a remote receiver tosummon an operator that may not be in visual or auditory range of thethawing device. In some embodiments the alert signals are terminated bythe removal of the sample vial from the warming block. Optionally, thesystem may automatically disengage one or more heating surfaces from thecontainer to automatically reduce the heating of the container. In someembodiments, the system may be configured to adjust one or more heatingsurfaces in contact with the container to maintain or hold thetemperature of the sample at a specific temperature after the end of thethawing or at the desired end point.

In some aspects of the present invention, a method for thawing a samplein a sample container is provided. The method may include the step ofheating a warming block and receiving the sample container within thewarming block. Thereafter, temperature measurements may be taken fromthe sample container and/or of the sample. A trigger point may bedetermined for using a time interval value for the remainder of theprocess. For example, a thaw start time (e.g., start of phase change)may be the trigger point for starting a time interval that identifiesthe end of the thawing process. The thaw start time may be determinedbased on the temperature measurements. Information on the sample and/orsample container may be received. A thaw completion time may bedetermined in-part on the thaw start time. The thaw completion time maybe adjusted per the received sample and/or sample container information.A signal may be provided for alerting a user to the thaw completiontime.

As the prediction of the thawing duration may be greatly facilitated bydepending only on a uniform starting temperature, a uniform warmingblock temperature, and a uniform sample vial configuration, in otheraspects of the present invention, devices and methods for equilibratinga sample vial to a standard starting ultra-cold temperature way pointwhile eliminating any direct contact of the sample vial with dry ice areprovided. In some embodiments of the invention, an insulating containerin which solid carbon dioxide or dry ice may be placed and upon whichmay be place or embedded a thermally conductive container for the samplevials is provided. In other aspects of the present invention, methodsare provided for retrieving a cryogenically preserved sample specimenvial from cryogenic storage at cryogenic temperatures andre-equilibrating the sample vial to an ultra-cold standard temperatureway point prior to initiation of the thawing process whereupon,following transfer of the equilibrated sample to a warm block ofstandard and uniform temperature, the duration of the thawing processmay be predicted on the basis of a known time constant exclusively. Inother aspects of the invention, methods are provide wherein thepreviously described method is applied, but also enhanced by additionalthaw time prediction capabilities derived from computational analysis ofsample vial external surface thermometric data.

In further embodiments of the present invention, a device may beprovided for the conversion from a solid state to liquid state of asample contained in a vessel. The device may include a pliable solidmaterial forming a receptacle for receiving the vessel and a heater forheating the pliable solid material to a temperature higher than amelting point of the sample. The pliable solid material may beinterposed between the vessel and a non-pliable solid material when thevessel is received within the receptacle formed by the pliable solidmaterial. The non-pliable solid material may comprise a material with athermal conductivity between 10 Watts per meter-Kelvin and 410 Watts permeter-Kelvin, with a typical thermal conductivity between 100 and 300Watts per meter-Kelvin, and in some embodiments a thermal conductivitybetween 150 and 180 Watts per meter-Kelvin. Materials with thermalconductivities in this range may exhibit a material hardness greaterthan a Shore durometer scale D value of 75 such that even whenmanufactured to close tolerances to mate with the vessel surface, minuteair gaps may be present in the interface between the vessel and thesolid receptacle, thereby introducing interruptions in the thermalconduction path across the material interface that may introduce avariance in thermal resistivity that is unpredictable in severity andfrequency of occurrence. In addition, although the general shape anddimensions of cryogenic storage vessels may be similar, variation bymanufacturing source is to be expected. Therefore, an interposition of athin layer of compliant material between the sample vessel and the solidmaterial receptacle can eliminate or substantially reduce the size andnumber of the air gaps and provide a uniform pathway through whichthermal energy may be transferred from the solid material to the samplevessel contents. An example of a pliable material would include, withoutlimitation, the thermally conductive pliable material sold commerciallyas Gap Pad VO soft by The Berquist Company, the material having a Shore00 hardness rating of 25 as determined by the ASTM D2240 testspecification. As elimination of air gaps between a solid materialreceiver and the sample vessel wall may require only a thin layer ofpliable material, a typical thickness, without limitation, of 0.5 mm to2 mm may be sufficient to insure adequate thermal contact, however aspliable materials may exhibit thermal conductivities that are low whencompared to solid materials, pliable materials with a thermalconductivity greater than 0.01 Watts per meter-Kelvin may be applied,while a typical pliable material may have a thermal conductivity greaterthan 0.1 Watts per meter-Kelvin, while in some embodiments the pliablematerial will have a thermal conductivity of greater than 0.5 Watts permeter-Kelvin.

The pliable material and the non-pliable material may be permanentlybonded together. Optionally, the pliable material and the non-pliablematerial may be removably bonded together.

In some embodiments, the pliable and the non-pliable material may besegmented into two or more segments and the segments may be joined by amechanical linkage that may move the segments into an open configurationfor receiving or releasing the vessel and a closed configuration forforming the receptacle and thawing the vessel. The pliable material maybe selectively placed in contact with the vessel when transitioning thesegments from the open configuration to the closed configuration orremoved from contact with the vessel when transitioning the segmentsfrom the closed configuration to the open configuration.

A vessel sensor may be provided for detecting the presence of the vesselbetween the segments of the pliable material when the segments are inthe open configuration and closed configuration.

A micro-controller may be provided for controlling the mechanicallinkage. When the segments are in the open configuration, themicro-controller may be configured to detect a placement of the vesselat a position between the segments while the segments are in the openconfiguration. The micro-controller may also be configured to deliver acontrol signal to actuate the mechanical linkage to move the segmentstoward the closed configuration to contact the vessel with the pliablematerial of the segments upon insertion of the vessel into the positionbetween the open segments.

When the segments are in the closed configuration and thawing thevessel, the micro-controller may be configured to interrupt thawing ofthe vessel by delivering a control signal to actuate the mechanicallinkage to move the segments toward the open configuration such that thepliable material of the segments do not contact the vessel.

In some embodiments, the non-pliable material may be heated by theheater.

A temperature sensor may be provided and may be fixed in the non-pliablesolid. The temperature sensor may be thermally insulated from thenon-pliable solid and may be held in contact with the vessel at acontact location such that a temperature signal reported by thetemperature sensor may be associated with a temperature of an exteriorsurface of the vessel at the contact location.

In some embodiments, the start of the phase change of a thawing samplemay be determined by algorithmic analysis of the temperature data fromone or more of the temperature sensors operably coupled with the vesselat a location below the top level of the sample contained therein.

The heating of the pliable material may cause radial heating of thevessel to achieve a thaw time that is predominantly independent of avessel fill level.

In further embodiments of the present invention, a method of thawingsample within a vessel may be provided. The method may include receivinga temperature data feed from a temperature sensor operably coupled withthe vessel (e.g., in direct contact with an exterior surface of thevessel at a location along the exterior surface of the vessel that isbelow a top level of the sample within the vessel, an infraredtemperature sensor, or the like), and identifying a start of a solid toliquid phase change of the sample contained in the vessel by processingthe temperature data feed. A thaw end time may be calculated based onthe start of the solid to liquid phase change of the sample contained inthe vessel. A signal may be outputted to interrupt thawing of the samplecontained in the vessel at the calculated thaw time.

In further embodiments, another method of thawing a sample within avessel may be provided. The method may include equilibrating the sampleand vessel to an intermediate temperature. The intermediate temperaturemay be below a sample melting temperature. Thereafter, the sides of thevessel may be contacted with a solid material mass that is maintained ata thawing temperature. The thawing temperature may be greater than 5degrees above the melting temperature of the sample plus or minus twodegrees. A thaw rate that is as rapid as possible may be desired tominimize the damage from ice recrystallization during the thaw process.A reduction in the thaw interval is favored by an increase in thetemperature of the vessel receptacle, however as certain vessel shapessuch as cylindrical formats may be associated with solid samplethicknesses greater than 1 centimeter in diameter, melting of solidmaterials contained therein will be accompanied by temperature gradientswith a higher temperature at the vessel interior wall, decreasing inmagnitude to the solid material remnant temperature toward the center.While liquid sample temperature transients due to temperature gradientsincurred by a 37° C. bath thaw of a standard 1.8 ml cryogenic vial donot seem to impact the viability of the majority of established celllines, the data set is not comprehensive and not reliably applicable tofresh cell isolates and primary cultures, and unfavorable results havebeen observed for some cryopreservation fluids at temperatures as low as5° C. Therefore, the optimal thawing rate for a given cell source orviral stock may be case specific, however receptacle temperatures areexpected to range from −1° C. to 100° C., typically from 20° C. to 55°C., and in some embodiments, 37° C. to 50° C. A completion of a phasechange of the sample contained in the vessel may be predicted bycalculating a time interval for a duration of the phase change based onthe equilibrating temperature and the heating temperature. A signal maybe outputted to interrupt thawing of the sample contained in the vesselat the predicted completion of the phase change of the sample containedin the vessel.

The intermediate temperature may be between −78° C. to −70° C. Thesample may be equilibrated to the intermediate temperature range byplacing the sample vessel into a receiving container that is in contactwith solid carbon dioxide. The receiving container and the solid carbondioxide may be surrounded on sides and a bottom by insulation. In someembodiments, the insulation comprises a polymer foam material including,but not limited to polyethylene foam, polyurethane foam, polyvinyl foam,polystyrene foam, and combination blends thereof. In some embodiments,the insulation comprises foam material exclusively while in otherembodiments, the insulation comprises a hard interior and exterior shellfilled with a reaction-in-mold foam such as polyurethane. In otherembodiments, the insulation comprises a stainless steel vacuum canister

In some embodiments, the intermediate temperature may be between −196 to−180 degrees Celsius. The sample may be equilibrated to the intermediatetemperature by placing the sample vessel into a receiving container thatis in direct contact with liquid nitrogen. The receiving container andthe liquid nitrogen may be surrounded on sides and a bottom byinsulation.

In some embodiments, the method may further include receiving a datafeed from a temperature sensor that is held in contact with an exteriorsurface of the vessel such that a temperature signal reported by thetemperature sensor is associated with a temperature of the exteriorsurface of the vessel, while in other embodiments the temperature signalis reported by an infra-red sensor that is not in direct contact withthe sample vessel and is receiving infra-red emission signals from thevessel contents through an optically-transmissive vessel wall or fromthe vessel wall directly. The calculated time interval for the phasechange duration may be adjusted based on the received temperature sensordata feed.

The invention will be better understood on reading the followingdescription and examining the figures that accompany it. These figuresare provided by way of illustration only and are in no way limiting onthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a model thawing system for a typical cryogenic storagevial that is use in describing the thermal energy flow pattern and vialtemperature detection methods.

FIG. 1B shows a conceptualized graphic of the temperature decrease fromthe warming block temperature to the solid sample phase under acondition of dynamic thermal energy flow.

FIG. 1C shows a cross-section perspective of an embodiment of a vialsurface temperature detection system.

FIG. 2 shows an embodiment of a vial temperature equilibration devicewith dry ice refrigerant that equilibrates a vial temperature toapproximately −77° C.

FIG. 3 shows a dimensioned drawing of the device described in FIG. 2.

FIG. 4 shows a second embodiment of a vial temperature adjustmentdevice.

FIG. 5 shows a dimensioned drawing of the device described in FIG. 4.

FIG. 6, graph A, shows a graphic display of the cooling andtemperature-holding duration of the embodiment described in FIGS. 2 and3. Graph B shows the uniformity in the temperature transition of samplevial contents when transferred from liquid nitrogen to thetemperature-equilibrated device described in FIGS. 2 and 3.

FIG. 7 shows an embodiment of a split-block vial thawing device.

FIG. 8 shows an exploded view of the device shown in FIG. 7.

FIG. 9, part A shows a graphic display of temperature data collectedwith a thermocouple located in an orientation coincident with thecentral axis of the sample vial at mid-depth in the sample duringmultiple thawing events in the device described in FIGS. 7 and 8.

FIG. 9 part B shows a graphic display of temperature data collected witha thermocouple located at the same depth as part A in an orientationparallel to the central axis of the sample vial near the interior wallof the same vial used to generate the data in part A.

FIG. 10 shows a series of temperature change data profiles from athermocouple positioned near the inner wall of a thawing vial as in FIG.9, part B, when the vial is placed into a 37° C. water bath or placedinto a warming block of the type described in FIGS. 7 and 8.

FIG. 11 shows an additional series of temperature change data profilesfrom a thermocouple positioned near the inner wall of a thawing vialwhen the vial is placed into a 37° C. water bath and into a 45° C.warming block of the type described in FIGS. 7 and 8.

FIG. 12 shows a graphic representation of the heat conduction pathwaysand heat flow rates into a storage vial at two different levels ofsample loading.

FIG. 13 shows a series of temperature change data profiles from athermocouple positioned near the inner wall of a vial when the vial isplaced into s 45° C. split warming block under conditions where the vialis filled with 0.5 ml of test solution and where the vial is filled with1.0 ml of test solution.

FIG. 14 shows temperature profile collected by a thermocouple that isplaced in contact with the exterior wall of a thawing sample vial asdescribed in FIG. 1. The warming block used was a split-block model andthe initial vial temperature was −77° C.

FIG. 15A, part A shows a graph of the time constant value output from anequation using the external vial temperature data shown in FIG. 14 asinput. Also shown is a graph of the same equation using as input valuesa linear time invariant (LTI) equation with variables adjusted to matchthe solid phase portion of the data in the graph shown in FIG. 14.

FIG. 15B shows a graphic, Part A, of a linear time invariant (LTI)lumped system analysis (LSA) curve with variable parameters adjusted tooverlay the solid-phase portion of a temperature-time plot using datacollected from a thermocouple that is placed in contact with theexterior wall of a thawing sample vial as described in FIG. 1. Part B ofFIG. 15 shows a temperature time graph of the difference between theoutput of the fitted LSA equation output in part A and the actualtemperature data collected for that time point. The graph indicates thetime point where melting of the sample begins, as determined by the plotin graph B exceeding a selected pre-set value limit of 0.2.

FIG. 16 shows the dimensions of three representative sample vials thatmay, without limitation, be thawed using the instant invention. Thevials include (A) a screw-cap cryo-storage vial with a nominal capacityof 1.8 ml, (B) a septum-cap vial with a nominal capacity of 2 ml, and(C) a septum-cap vial with a nominal capacity of 10 ml.

FIG. 17 shows the exterior of a representative embodiment of theinvention.

FIG. 18 shows two perspectives of a representative embodiment of theinterior mechanism of the device shown in FIG. 17.

FIG. 19 shows the overall dimensions of the embodiments of the inventionshown in FIGS. 17 and 18.

FIG. 20 shows an exploded view of the embodiment shown in FIG. 18.

FIG. 21 shows the first two steps (step 0 and step 1) of the thawingcycle of the embodiment shown in FIGS. 17 through 20. In the two-stepillustrations (and in FIGS. 22 and 23), the front-most heater block halfis shown removed to better reveal the mechanism positions and actionduring the six cycle steps.

FIG. 22 shows the subsequent two steps, step 2 and step 3, of the thawcycle to those illustrated in FIG. 21.

FIG. 23 shows the subsequent two steps, step 4 and step 5, of the thawcycle to those illustrated in FIG. 22.

FIG. 24 shows a time-temperature plot of two series of thawing profiles,the first series in which the jaws of the 45° C. warming block wereopened 150 seconds into the thaw and the vial was left in-situ in thewarming block, and a second series in which the jaws of the warmingblock were opened 150 seconds into the thaw and the vial was removedfrom the warming block and held at room temperature. A comparative tracefrom a vial that was left in the 45° C. block with the jaws closed forapproximately 5 minutes is also shown.

DETAILED DESCRIPTION

The subject matter of embodiments of the present invention is describedhere with specificity, but this description is not necessarily intendedto limit the scope of the claims. The claimed subject matter may beembodied in other ways, may include different elements or steps, and maybe used in conjunction with other existing or future technologies. Thisdescription should not be interpreted as implying any particular orderor arrangement among or between various steps or elements except whenthe order of individual steps or arrangement of elements is explicitlydescribed.

In some embodiments of the invention, direct liquid contact with thesample vial exterior may be eliminated, as would occur with a partialsubmersion of the sample in a water bath. As such, in many embodimentsof the invention the exterior surface of the sample vial, or in somecases the sample vial exterior plus laminations such as labels orshrink-wrap sleeves, will be in contact only with solid materials. Insome embodiments the solid material in contact with the vial exterior isa homogenous solid, while in other cases, the solid material is acomplex material. In some embodiments, the solid material has a thermalconductivity of greater than 0.2 watts per meter-kelvin. In someembodiments the solid material comprises aluminum, copper, zinc,magnesium, titanium, iron, chromium, nickel, carbon, and alloys of thesame elements. In some embodiments, the solid material may comprise asynthetic material such as a polymer or ceramic. In other embodiments,the solid material may comprise a synthetic thermally-conductive pliablematerial such as, but without limitation, the silicone polymer foamprovided by The Berquist Company under the brand name Gap Pad VO. Insome embodiments the solid material comprises combinations of materials,for example and without limitation, a conductive pliable material and ametal alloy. In some embodiments the solid material in contact with thesample vial exterior is a polymer shell or tank containing a liquidfilling while in other embodiment, the solid material comprises apolymer shell or tank containing a liquid filling that comprises athermally conductive lining of pliable material that contacts the samplevial contained therein. In some embodiments the liquid in the polymershell is water or aqueous solutions while in other embodiments theliquid is an oil or a liquid organic material. In other embodiments theshell is filled with a wax that is liquid at some temperatures whilesolid at other temperatures.

In some embodiments, the sample vial or a portion of the sample vial isin continuous contact with the solid material on the circumference ofthe vial while in other embodiments, the solid material contacts thevial intermittently. In some embodiments, the solid material comprises arecess or cavity which closely matches the outer surface of the samplevial for the purpose of receiving the sample vial such that the samplevial is partially contained within the solid material in direct andclose contact. In some embodiments, the container cavity comprises oneor more sides and a floor, while in other embodiments, the containercomprises only one or more sides. In other embodiments, the solidmaterial containing the sample vial is segmented to facilitate theinsertion and removal of the sample vial from the material and tointerrupt the thermal conduction pathway between the solid material andthe sample vial. In some embodiments, the solid material segments of thecontainer are relationally constrained such that when separated tofacilitate insertion or removal of the sample vial or to interrupt thethermal conduction between the solid material segments and the samplevial, the segments can be easily reassembled into a closely joinedconfiguration. Without limitations, in some embodiments, the segmentsare joined by slide mechanisms, hinge mechanisms, track mechanisms,hydraulic or pneumatic pistons, rails, kinematic linkages, pin andgroove linkages, electro-magnetic, or magnetic interface. In otherembodiments, the segments are, without limitations, actively separatedor joined by electric motors, solenoid activators, pneumatic orhydraulic actuators, linear actuators, either directly acting on thesegments or by gear systems, kinematic linkages, cam systems, pushrods,cable system, and screw mechanisms.

In some embodiments, the solid material container for the sample vialcomprises one or more heater elements for the purpose of increasing thetemperature of the solid material such that when a sample vial is placedinto the receiving cavity, thermal energy will migrate into the samplevial (from here forward referred to as “warming blocks”). In somewarming blocks the heater elements are electrically resistive heaterswhile in other warming blocks, the heater elements are thermoelectricelement heaters. In some embodiments, the warming block may bealternatively heated and cooled by a thermoelectric element. In someembodiments, the warming block comprises one or more temperature sensorsthat can detect the temperature of the block and provide an analog ordigital signal to a microcontroller that is configured to interpret thethermometric signal and thereby regulate the power level or duty cyclesupplied to the heater element in order to maintain the temperature ofthe warming block at the desired temperature.

In some embodiments, the warming block comprises one or more temperaturesensors that are thermally isolated from the warming block material butare in contact with the exterior surface of the sample vial such thatthe temperature of the vial at the surface may be ascertained andtracked over time (from here forward referred to as the “vial sensor”).In some embodiments the vial sensor is a thermocouple while in otherembodiments, the vial sensor is a thermistor or an RTD sensor. In otherembodiments, the vial temperature is sensed by a non-contact infra-redsensor.

When a cylindrical sample vial that has been equilibrated to a lowtemperature, for example and without limitation, −77° C., is insertedinto a warming block that has been equilibrated to a higher temperature,for example 45° C., a process of thermal energy redistribution willcommence that will eventually bring the combined masses to a commontemperature. If the warming block temperature is actively maintained,for example at 45° C., then the temperature of the combined masses willin time equilibrate at a temperature of 45° C. The thermal energyredistribution pattern may be considered to be a migration or flow ofthermal energy in a radial pattern toward the central axis of thecombined mass.

Now referring to FIG. 1A part A, a front cross-section view, and B, atop cross-section view, a representative model of a warming block isshown. In this figure, an aluminum alloy cylindrical container 120comprises a central cavity wherein a lining of thermally conductivepliable material 125 surrounds the vertical wall of the cavity exceptwhere an opening allows a thermistor temperature sensor 130 to protrudeinto the cavity. In this figure, the cavity is occupied by a sample vialtube 110 that is sealed with a screw cap 115 the combination of whichisolates the sample contents comprised of a liquid phase 140 and a solidphase 135. The vial interior also comprises a gas phase volume 145. Thethermometric sensor 130 is in direct contact with the exterior surfaceof the sample vial tube such that the temperature measured is thetemperature of the exterior surface of the vial. Segmentation lines 150bisect the aluminum alloy container and the thermally conductive pliablematerial. The aggregate of the components shown in FIG. 1A may beconsidered collectively to represent a system in reference to thethermodynamic illustrations to follow.

FIG. 1B shows the same graphic A as in FIGS. 1A and 1 s used to markspecific material boundaries at the indicated radii. The boundary lines180 to 185 define a region comprised of the aluminum alloy, lines 175 to180 define a region comprised of thermally conductive pliable material,lines 170 to 175 define a region comprised of sample vial material sucha polypropylene, polyethylene, or blends of polypropylene, polyethyleneand additional plastic materials, lines 165 to 170 define a region ofliquid sample phase, and lines 160 to 165 define a region of solidsample phase. As the model shown contains both liquid and solid phase,the state of the sample shown is during the melting or phase transitionprocess. When the system shown is in the process of thermal energyredistribution, dynamic temperature gradients are established within thevarious materials such that the temperatures at the various materialboundaries become dependent on the thermal resistivity of the materials.As represented in FIG. 1B, part B, the temperature with in the aluminummaterial is nearly uniform due to the low thermal resistance or highthermal conductivity (approximately 170 W/m-K) of the material. With thedynamic heat flow, however, a temperature drop is established across theregion occupied by the thermally conductive pliable material (175 to180) due to the relatively higher thermal resistance or relatively lowthermal conductivity of the pliable material (approximately 0.8 W/m-K).A greater relative temperature drop will occur crossing the vial wallmaterial (170 to 175) which has the greatest thermal resistance orlowest thermal conductivity of the system (approximately 0.2 W/m-K).Crossing the liquid sample material (165 to 170) a more shallowtemperature drop will occur as the thermal resistance is similar invalue to that of the thermally conductive foam (thermal conductivity ofapproximately 0.6 W/m-K), and the temperature drop across the solidsample material will be more shallow due to the lower thermal resistancewhen compared to the liquid phase (thermal conductivity of approximately2 w/m-K). As the magnitude of the temperature decrease across thevarious materials increases with the magnitude of the difference in thewarming block temperature (T₁) and the sample temperature (T₂), thetemperature decreases across the various materials will be greatest inmagnitude shortly after the sample vial is first inserted into thewarming block and least in magnitude as the system approaches anequilibrium temperature. At any given time during the period of thermalenergy migration, the relative temperature decreases across the variousmaterials comprising the system will be a function of the thermalresistance of the various materials, the value of which does not changethroughout the process, therefore the temperature at any one of thematerial boundaries can be considered to be proportional to thetemperature at the other material boundaries, with the exception of theliquid-solid phase 165 (r₁) which will be subject to movement throughoutthe phase transition thereby changing the radius value and temperaturedepending on the location of the boundary in the system. Therefore atime-temperature trace at the conductive pliable material boundary 175(r₃) can be an accurate proportional representation of thetime-temperature trace at the vial wall-liquid boundary and a closeapproximation of the average temperature of the liquid phase. Therefore,by monitoring the external vial temperature during the thawing process,the time-temperature profile of the vial contents can be closelyapproximated, thereby allowing the progress of the sample thawingprocess to be determined non-invasively.

The amount of thermal energy required to raise the temperature of asample vial and the contents from one temperature to a secondtemperature is dependent only on the heat capacity of the sample vialand the sample mass contained therein. Therefore if the material massesand the hence the amount of heat required to achieve the temperaturetransition do not change, and the start temperature of the warmer blockand the start temperature of the sample vial are consistent, the sametime temperature profile may be expected upon repeated freeze-thawcycles of the same sample. If the sample vial dimensions, vial materialsand mass, and sample payload mass and composition are uniform fromsample to sample, the time-temperature profiles obtained should beidentical regardless of whether the same sample is repeatedly cycledthrough a freeze-thaw or another sample is subjected to the sameprocess. Therefore, the inclusion of a step or device for equilibratingall samples to a uniform starting temperature, and an accurate anduniform warming block temperature will allow the prediction of thethawing process duration to be made exclusively on the basis of priorexperience.

Now referring to FIG. 1C, a detailed cross-section of an embodiment ofthe sample vessel surface temperature sensor (130 in FIGS. 1A and 1B) isshown. In this figure, a glass-encased thermistor bulb 132 is in directcontact with a thermally conductive coupler 131. In some embodiments,the coupler comprises a highly conductive material such as, but notlimited to, aluminum, silver, copper or alloys comprising aluminum,silver or copper. The coupler 131 is in direct contact with the outsidesurface of the sample vessel wall 110. A semi-rigid foam insulatorsleeve 133 holds the coupler 131 against the vessel surface, and is heldunder compression by a ram piston 134 that comprises an insulatingmaterial. In some embodiments the ram material is, without limitation,an acetal or phenolic polymer. The ram piston is placed undercompression by a spring 136 that is captured between the sliding ram 134and a screw plug 137 that is fixed in a threaded access hole through thewarming block. A through hole in the screw plug 137 allows the passageof the thermistor lead wires 139 to the exterior of the block. A gap inthe conductive foam 125 allows direct contact between the coupler 131and the vessel wall 110 and limits direct thermal energy influx from thewarming block 120. The thermal energy path created from the warmingblock 120 through ram piston 134, insulating sleeve 133 and coupler 131to the vessel wall 110 creates a thermal resistance stack such that byselection of thermally resistive materials for the piston 134 and sleeve133 and a thermally conductive material for the coupler 131, thetemperature of the coupler, and hence the thermistor bulb 132, isclosely coupled to the temperature of the vessel exterior surface 110and therefore the temperature reported by the thermistor closely followsthe temperature of the vessel surface. In some embodiments a thin layerof a pliable material (not shown), with an approximate thickness between0.005 inches and 0.04 inches may be bonded to the coupler 131 andinterposed between the coupler 131 and the vessel wall 110 to augmentthermal conduction. In other embodiments, the thermistor assembly (131,132, 133, 134, 136, 137, 139) is replaced by an infra-red thermalsensor. As materials used in the construction of cryogenic vessels maycomprise materials that are optically transparent to infra-red light,the temperature of the vessel contents may be measured directly byinfra-red emission. Where the vessel material is optically opaque toinfra-red light, or the vessel may comprise an optically opaque label,the surface temperature may be measured by an infra-red sensor and theprogression of the phase change from solid to liquid may be detected.The infra-red sensor has additional advantages in that physical contactbetween the sensor and the vessel is not required and thereforepotential problems associated with sensor pressure on the vessel wall,variance in thermal conduction in the thermal sensing pathway, andpotential sensor damage due to improper insertion of a sample vessel areeliminated.

Now referring to FIG. 2, a device is shown that may be used toequilibrate sample vials to a reference temperature (or intermediatetemperature). In the figure, a sample vial receiver comprises arectangular upper block of solid material 215 and a horizontal flange230 that is mated to upper block forming a sample receiver block. Theupper block 215 comprises one or more recesses 220 that are sufficientdiameter and depth to receive and surround the sample vial such that thetop of the sample contained within the vial is below the top surface ofthe block. In some embodiments, the receiver block comprises one or morerecesses on the sidewall of the upper block 240 to assist in the gripsecurity of the invention. In some embodiments, the flange 230 and theupper block 215 interface as an uninterrupted continuum of the materialfrom which the parts are made, while in other embodiment the upper block215 and the flange 230 are separate pieces that are joined, withoutlimitations, by mechanical fasteners, adhesive bonds, magneticfasteners, or weldements. In some embodiments, the upper block comprisesa hole (not shown) into which a thermometric sensor may be inserted andsecured. In some embodiments, the sample receiver block is constructedfrom a metal. In some embodiments, the metal comprises aluminum, copper,magnesium, zinc, titanium, iron, chromium, nickel or alloys of thesemetal elements. In some embodiments, the receiver block is surrounded onthe sides and bottom by an insulating container 210 that has a cavity245 with an interior height that is greater than the height of thereceiver block plus 1 inch. In some embodiments, the insulatingcontainer comprises an insulating foam material. In some embodiments theinsulating foam material comprises polyethylene, polyurethane, orpolystyrene, while in other embodiments, the insulating materialcomprises a blend of materials such as a polyethylene polymer blend. Insome embodiments, the insulating container comprises a cover (notshown). The receiver block is positioned in the insulating containersuch that as layer of solid carbon dioxide or dry ice 225 is positionedunder and above the lateral surface of the flange 230. Although solidcarbon dioxide in contact with a surface that is above the temperatureof −78.5° C. will sublime, thereby forming a gap between the solidcarbon dioxide and the surface and interrupting the direct contactthermal energy conduction pathway between the materials, in agravitational field, the dry ice will remain in direct contact with theundersurface of the receiver block and the upper surface of the receiverblock lateral flanges. In embodiments the receiver block, withoutlimitation, comprises a solid material has a thermal conductivitygreater than 16 W/m-K such as aluminum alloy. The receiver block shownin FIG. 2 will maintain a steady temperature of −77° C. in an open-topconfiguration. As the interior walls of the container exceed the heightof the receiver block by at least one inch, the amount of dry icebeneath the receiver block may be limited such that the entirety of asample vial placed into a receiver well will be positioned below the topsurface of the insulating container, thereby holding the sample in awell of cold gas, and insulating the upper portion of the vial from theenvironmental temperature. In this configuration, vial temperatures, asmeasured with an internal thermocouple can be equilibrated to and heldat a reference temperature of −77° C. As such, the reference temperaturedevice shown in FIG. 2 can be used to provide a standard startingtemperature for the sample thawing process that will allow theprediction of the thaw process status based exclusively on the durationof the thaw process.

Now referring to FIG. 3, the overall dimensions of the device shown inFIG. 2 is shown. The embodiment has an outside width of approximately 7inches, a width of approximately 5.5 inches and a depth of approximately3.5 inches. The internal cavity has a length of approximately 5.25inches, a width of approximately 3.6 inches, and a depth ofapproximately 2.8 inches. The receiver block has a length ofapproximately 5 inches, a width of approximately 3.4 inches and a heightof approximately 1.35 inches. The sample vial receiver wells of thereceiver block have a diameter of approximately 0.55 inches and a depthof approximately 1.1 inches. In some embodiments, the wells of thereceiver block comprise a passage in the floor of the wells that extendsto the undersurface of the receiver block so that the receiver block maybe used efficiently with a liquid refrigerant such as liquid nitrogen.In FIG. 3 the passage way has a diameter of approximately 0.2 inches.Although the dimensions of the sample receiver wells shown are forreceiving a standard laboratory screw-cap cryovial, the dimensions,spacing and number of the sample receiving wells may be adjusted toaccommodate sample vials of other dimensions. In some embodiments, thediameter and depth of the wells may be adjusted to provide a go no-gogauge for the sample vial by which a user may determine if the vialintended for the thawing process is too large for the thawing apparatusor is too small to be used properly with the same.

Now referring to FIG. 4, a second embodiment of a temperatureequilibration device is shown. In this embodiment, a circular receiverblock 430 is shown comprising a radial distribution of sample vialreceiver wells 440. In some embodiments, the receiver block may comprisea central well 450 that may be provide an additional vial receiver well,be used as a through hole by which to assess the presence of dry icebeneath the receiver block, or provide a gauge for the purpose ofconfirming the appropriate vial dimensions that are compatible with thethawing device. The receiver block 430 is situated within the internalcavity of an insulating container 410. In some embodiments, the receiverblock 430 comprises a hole into which a temperature sensor may beinserted and secured (not shown). In some embodiments, the insulatingcontainer 410 comprises internal extensions of the cavity wall 420 thatsupport or limit the movement of the receiver block 430, while in otherembodiments, the internal wall of the insulation housing 410 is acylindrical shape without extensions. In some embodiments, the receiverblock and the insulating container comprise the same materials describedfor the embodiments presented in FIGS. 2 and 3. In some embodiments, thereceiver block 430 comprises a disc-shaped flange attached at the bottomsurface of the receiver block (not shown) while in other embodiments thereceiver block comprises the upper block only.

Now referring to FIG. 5, the overall dimensions of the device describedin FIG. 4 are shown. The insulating container has an outside diameter ofapproximately 5.5 inches and a height of approximately 3.5 inches withan internal cavity diameter of approximately 2 inches and a depth ofapproximately 2.8 inches. The receiver block has an outside diameter ofapproximately 2.5 inches and a height of approximately 1.4 inches. Thevial receiver wells of the receiver block have a diameter ofapproximately 0.51 inches and a depth of approximately 1.15 inches. Thecentral cavity has a diameter of approximately 0.7 inches and in theembodiment shown extends through to the undersurface of the block. Insome embodiments, the vial receiver wells of the receiver block comprisea passage 470 extending through the floor of the well to allow floodingof the well when the receiver rack is used with a liquid refrigerantsuch as liquid nitrogen. In some embodiments the passage has a diameterof approximately 0.2 inches. In other embodiments, the sample vialreceiver well floor is solid and does not comprise a passageway.

Now referring to FIG. 6, part A, a data graph of the temperature of areceiver block as described in FIGS. 2 and 3 is shown. The temperaturemeasurements were collected by a thermocouple sensor that was positionedinto a receiver hole drilled into the vial receiver block to a depth of0.5 inches. The receiver block was placed upon an approximately 0.75inch thick layer of dry ice, and additional dry ice was placed over theflange portion of the receiver block to a level equal to the top of thereceiver. The receiver was allowed to temperature equilibrate. As seenin the graph, the receiver block reached a temperature of −77° C. inapproximately 5 minutes and held the temperature for over 7 hours untilthe dry ice was exhausted. During the 7-hour interval a sample vialcontaining 90 percent buffered saline and 10% dimethyl sulfoxide in avolume of 1 ml was configured with a thermocouple temperature sensorheld in an axial orientation with the thermocouple sensor positionedmid-height in the sample liquid. The sample vial was then equilibratedto a temperature of −194° C. in liquid nitrogen, then transferred to the−77° C. equilibration block. As shown in part B of FIG. 6, thetemperature of the vial contents equilibrated to the −77° C. temperaturewithin an interval of approximately 10 minutes. Following repeatedcycles of thawing, re-equilibration in liquid nitrogen and transfer tothe −77° C. receiver block, the temperature profiles of the samplecontents are highly repeatable. Using this simple equilibration deviceand method, a sample stored at cryogenic temperature may be retrievedfrom archival storage and be rapidly equilibrated to a steadytemperature of −77° C. The sample may then be stored for an extendedperiod of up to 7 hours or longer if the dry ice refrigerant isreplenished. The receiver block at −77° C. provides a highlyreproducible temperature start point for a thawing process, allowing thethaw time of a sample to be accurately predicted following placementinto a warming block that has been equilibrated to the appropriatetemperature. In addition, the receiver block prevents direct contact ofthe sample with the dry ice refrigerant. As some vial designs comprise askirt extension on the undersurface (see FIG. 16, vial A), directinsertion of these vial into dry ice will capture dry ice in theunderside recesses and if subsequently inserted into a warming blockwill experience a significant change in the thaw time due to theadditional heat influx required to change the dry ice to the gas phase.Therefore the use of a receiver block that isolates the sample vialsfrom direct contact with the dry ice is preferable for thestandardization of the thawing process.

Now referring to FIG. 7, a standard warming block is shown. In thisembodiment, the warming block 710 is segmented by two right-angle planarinterfaces 770 and 780 to create an independent block segment 720. Thevertical segmentation plane passes through the center of a cylindricalsample vial receiving well coincident with the cylindrical axis of thereceiving well. The receiving well shown comprises a 1-degreecylindrical taper to match the taper of a standard screw-capped cryovial750 such as those available commercially from multiple vendors and beingcomprised of a material having a thermal conductivity of less than 2Watts/meter-K including, but not limited to, polypropylene,polyethylene, or blends of polypropylene, polyethylene and additionalplastic materials, plastic resins and resin blends, and glass. Thereceiving well walls comprise a 0.5 mm-thick layer of thermallyconductive material such as, but not limited to, thermally conductivefoam 760, with the innermost surface matched the surface of the standardscrew-capped cryovial such that when the cryovial is placed into thewell and the two receiver blocks close to near contact at the surface770, the cryovial surface and the conductive foam are in close andcomplete contact at all points. The movable sliding segment 720 isconfined to a linear horizontal motion by two push-rods (hidden in thisperspective) that are secured at the end by a push-bar 730. Byhorizontal actuation of the push-bar, the segments of the warming blockmay be separated to allow insertion or removal of the cryovial. It maybe noted that an embodiment that does not comprise the thermallyconductive foam can be constructed, however, perfectly matching thetaper of the cryovial is a difficult achievement and from manufacturerto manufacturer, variation in the angle of the taper and the diameter ofthe vial may be encountered. In addition, upon freezing, the aqueouscontents of the sample vial will expand with the potential to distortthe exterior surface of the cryovial. Further complications may arise inmating the receiver well surface to the vial exterior surface in thatthe vials may be unpredictably laminated with an identification label,therefore a compliant surface in the receiver well is essential touniform, complete and repeatable contact of the two surfaces as anydisruption in the physical contact will alter the thermal transfer byimposing additional thermal resistance at the location of thedisruption. Therefore, the thermally-conductive and compliant interface760 is preferred in some embodiments. The warming block 710 is heated byan electric resistance heater (hidden in this view) that is embedded inthe undersurface and powered by an electrical current. The temperatureof the warming block may be determined by a thermocouple sensor 740 isinserted into the warming block segment 710. The block segments 720 and710 are further joined by embedded magnet pairs both on the verticalinterface 770 and the horizontal interface, thereby assuring closethermal conductive contact of the two parts in addition to supplyingclamping pressure to the inserted vial. The embodiment parts 710, 720and 730 are constructed from an aluminum alloy. The thermally conductivefoam lining is constructed from a thermally-conductive siliconecomposition sold commercially by Laird Technologies under Tflex brand.

Now referring to FIG. 8, an exploded diagram of the embodiment shown inFIG. 7 is shown. In this diagram the L-shaped warming block 810 mateswith a rectangular block 815 at two interface planes, 811 and 812. Thetwo block segments are removably fastened on the vertical plane 811 bythe interface of two magnet pairs 830 that are received in block 810 inthe receiver cavities 835, and mirrored receiver holes on therectangular block 815 (not visible in the view). On the horizontal plane812, the two block segments 810 and 815 are joined by a single magnetpair in which one magnet 850 is embedded in the undersurface of block815 while two separate opposing magnets 840 are embedded in thehorizontal surface 812 in receiver holes 845. The single magnet 850 insliding block 815 may selectively interface with either of the twomagnets 840 by changing linear position along the axis defined by edge841. The magnet 840 centers are spaced 0.28 inches apart and allow theblock 815 to assume two stable positions, one being a position where thesliding block 815 is mated to block 810 at the 811 and 812 interfaces,and another where the two blocks are interfaced at plane 812 and with agap of approximately 0.27 inches between the two vertical block faces,thereby establishing an open and closed warming block states. Twothermally pliant conductive material linings 885 are laminated onto thetwo inside wall halves of the vial receiver well 857. The block segmentsare warmed by a resistance heater 855 that is embedded into theunderside of the block 810 in a wedge-shaped cavity and the heaterelement 855 is held in close thermal contact with the cavity walls bypressure from a wedge-shaped segment 860, the pressure on which may beadjusted by a force of a screw impinging on the back side of the wedge(not shown in this view). The heater is powered by an electrical currentthat is conducted through power wires 865. The temperature of thewarming block may be monitored by the thermocouple sensor 870 insertedinto the warming block 810 at the sensor receiver hole 875. The block815 is supported laterally by two push-rods 820 that are bridged by apush bar 825 at the distal ends. The push rods extend through block 810through access channels 826 which contact the push rods on the sidesonly and are machined in a vertical slot configuration to allow somedegree vertical freedom such that the block 815 is supported exclusivelyby the horizontal surface 812 of the block 810. The warming blockoperates by manual separation of the block segments 810 and 815 bypressing on the push bar 825. A sample vial is inserted into the openedreceiver cavity 857 and the jaws are closed by slight pressure on theblock 815 until the magnet 850 separates from the proximal magnet 845and re-aligns with the distal magnet 845 assisted by the added pull ofthe magnet pairs 830. In other embodiments, the warming block shown inFIG. 8 can be articulated and automated by active propulsion machineryincluding but not limited to motors, solenoid actuators, hydraulic andpneumatic actuators, and electro-magnets. The propulsion machinery maybe linked to the block segments, without limitation, by screw machines,kinematic linkages, hinges, cables, belts, chains, pin and slot, tracks,rails, slides linear and rotational bearings, cams, and gears. In someembodiments, the system shown in FIG. 8 may comprise more than onetemperature sensors by which the temperature of the warming block may bemonitored. In some embodiments, the heater block comprises one or moretemperature sensors that are thermally isolated from the warming blockbut are in contact with the surface of the sample vial. In someembodiments, the temperature sensors may comprise thermocouples,thermistors and RTD sensors. In other embodiments, the warming blockcomprises a microprocessor circuit board which receives warming blocktemperature feedback signals from the block sensors and regulates thepower supplied to the heaters to maintain the desired block temperature.In other embodiments the microprocessor board receives position sensordata from proximity sensors on the warming block to determine when theblock is open or closed. In other embodiments, the microprocessor boardactively opens and closes the warming block according to a statealgorithm that conducts the thawing process. In other embodiments, themicroprocessor receives thermometric signal data from sensors in contactwith the surface of the sample vial received into the warming block. Inother embodiments, the microprocessor makes determinations of thethawing status of the vial by algorithmic interpretation of thethermometric data. In some embodiments, the microprocessor boardcontrols a user interface that displays the status of the thawingprocess, alerts the users to fault conditions and signals the readinessof the warming block to receive a sample vial and initiate the thawingsequence.

In some embodiments, the size of the magnet pairs 830 and/or the fieldstrength of the magnet pairs may be used to adjust the clamping pressureof the conductive pliant material linings 885 on the vessel 880, therebychanging the thermal conduction between linings and the vessel. In otherembodiments the clamping pressure is provided by, without limitations,magnetic, electromagnetic, spring, pneumatic, hydraulic, or mechanicalforce, or any combination thereof.

Now referring to FIG. 9, part A, a series of time-temperature traces areshown from a thermocouple sensor positioned internally in a sample vialalong the central axis of the vial with the sensor bead at half-depth ina 1 ml sample comprising 90% buffered saline and 10% dimethyl sulfoxide.The vial was equilibrated to −194° C. in liquid nitrogen, then placedinto the equilibration device described in FIGS. 2 and 3 to equilibrateto −77° C. for 10 minutes. The vial was then transferred to the warmingblock device described in FIGS. 7 and 8 that was pre-equilibrated to 37°C. for 6 minutes while a data recorder collected the temperature traceat 10-second intervals. The freeze thaw cycle was repeated 6 times andthe time temperatures traces were plotted collectively. From the tracegrouping, it can be seen that the thawing profile is highly repeatableuntil approximately 3 minutes. Near the three-minute mark, the centralsolid remnant of the sample is confined as the axial thermocouple isstill embedded in the solid mass. As this solid remnant is released fromthe thermocouple sensor, the lower temperature solid is free to randomlyseparate from, contact, or intermittently contact the sensor, therebyintroducing artifacts into the data stream. The repeatability of thedata traces prior to the 3-minute point indicates that by starting at aconsistent sample temperature, and using a regulated temperature warmingblock, the progress of the thawing process can be closely predictedusing only experimentally-derived thaw interval time values. Therefore,in some embodiments a chronometric device, a temperature equilibrationdevice as shown in FIGS. 2 through 5, and a constant temperature warmblock as shown in FIGS. 7 and 8, are used to make an accurate predictionof the duration of the thaw process for a given sample vial. In otherembodiments the reduction of the phase change completion time value by aconstant will be used to terminate the thawing process while some solidphase is still remaining in the vial.

FIG. 9, part B shows a cluster of temperature traces from a thermocoupleplaced into the same sample vial such that the sensor lies near theinterior wall of the vial. The traces reproduce those observed in FIG.9, part A until approximately the one minute mark at which time thephase change begins. The temperature trace displays a higher trajectorythat that observed in part A, indicating that the temperature near theinner wall is warmer than at the center of the sample load. This outcomeis predicted by the complex thermal resistance pathway that the thermalenergy must traverse as described in FIG. 1B. It is also noted that nearthe 3 minute time mark, artifacts in the temperature trace cluster arepresent, but with less severe deviation from the average temperaturevalues than observed in FIG. 9, part A.

Now referring to FIG. 10, additional data was collected using the liquidnitrogen freezing and the −77° C. dry ice temperature equilibrium deviceused to collect the data in FIG. 9 with the thermocouple sensor situatedin the same position used in FIG. 9 part B, near the interior wall ofthe vial. In the data shown, however one cluster of three cycles wascollected using 37° C. warming block described in FIGS. 7 and 8, whilethe other cluster of three cycles was collected by partial submersion ofthe vial in a 37° C. water bath. Comparing the traces, it can be seenthat the vials that were thawed in the warming block (indicated as the“split block” in the figure), are thawing at a slower rate. Againreferring to the thermal energy flow model across a complex thermalresistance pathway as described in FIG. 1B, the result can be understoodas there would be a temperature drop across the conductive foam materialeffectively placing the sample vial in a lower temperature environmentthan would be experienced by the same vial in a 37° C. water bath.

Now referring to FIG. 11, the thawing series described in FIG. 10 wasrepeated using the identical systems with the exception that thetemperature of the warm block or split block was equilibrated to 45° C.prior to beginning the thaw series. In the time-temperature graphsshown, the two trace clusters are overlaid indicating that by elevatingthe warm block temperature, the temperature at the conductivefoam-sample vial interface can be elevated to 37° C., effectivelycreating a water bath-equivalent thaw using a solid warming block.

Now referring to FIG. 12 a cross-section diagram of two sample vials isshown to illustrate the load-volume independence of a thawing vialtemperature trace for a cylindrical sample vial. In the vial A where thecontents of the vial is greater, an identical thermal conduction pathwayexists at the position of both of the arrows in that the inner wall ofthe vial is in direct contact with the sample. In vial B, the samplevolume is reduced and therefore at the position of the upper arrow, theinterior wall of the vial does not contact the sample, therefore thermalenergy entering the vial at this location must either migrate downwardthrough the thermally resistant polymer of the vial wall or migratethrough the gas above the vial which has a thermal conductivityapproximately one-tenth that of the polymer vessel wall. Therefore, theamount of heat entering the sample contained within the vial isproportional to the amount of sample in the vial. This effect isexperimentally demonstrated in FIG. 13.

Now referring to FIG. 13, temperature trace clusters were generatedusing the same warm block thawing system and method described in FIG.11, with the exception that two different sample vial loads were usedfor the two thaw series. In one trace series the vials contained 1 ml ofthe test sample fluid and in the second series, the vials contained 1.5ml of the test sample fluid. As can be seen in the time-temperatureplots, the two series overlay indicating that the thaw profile isindependent of the vial fill volume. It may be noted for the discussionof FIG. 14 that the beginning of the phase change occurs atapproximately 50 seconds after the insertion of the vial into the warmblock and that the end of the phase change occurs at approximately 160seconds after the insertion of the vial into the warm block.

In the process of recovering viable biological samples from cryogenicstorage, the optimal recovery of live cells is favored by rapidtransition through the ultra-cold temperature region of −75° C. to theliquid state, as this practice will minimize the opportunity forinjurious intra-cellular ice crystal grown to occur. While an increasein the bath or warming block temperature will decrease the duration ofthe temperature transition by increasing the rate of thermal energyinflux into the sample vial, the experimental evidence shown in FIG. 9demonstrates that during the thawing process, the temperature of aportion of the sample will experience temperatures higher than thephase-transition temperature of the sample. Although the temperatureincrease in regions of the sample due to the dynamic heat influx aretransient, the toxicity of the cryoprotectants commonly included in thecryopreservation media for cell suspension cryopreservation increaseswith temperature, therefore reducing the thawing interval by increasingthe bath or warm block temperature has associated risks for a portion ofthe sample. To reduce the exposure of the thawed sample to elevatedtemperatures, a common practice for sample thawing includes thecessation of exposure of the sample vial to the elevated temperature ofthe water bath at a time when a small portion of the solid sample stillremains. This practice allows the still-solid remnant to absorb thermalenergy from the liquid portion of the sample, thereby equilibrating thethawed sample to a low temperature. The accurate assessment orprediction of the nearly complete phase change state during the thawingprocess is therefore necessary. During manual thawing of a sample vialin a water bath, a common practice includes the frequent visualassessment the sample state. As this practice requires the removal ofthe sample from the water bath, variation in the duration of the thawtime is imposed as the thermal contact between the vial and the waterbath heat source is frequently interrupted. Unless an alternativeassessment of the sample state is applied, a repeated visual inspectionof the sample would also require removal of the sample vial from a solidwarming block as well, and under such conditions, a standardized thawingmethodology could not be applicable. Intra-vial thermometry wouldprovide monitoring of the status of the phase change in a sample,however the introduction of a thermometric probe directly into a samplewould impose a very high risk of contamination. Therefore, in someembodiments of the instant invention, thermometric monitoring of theexternal surface temperature of the sample vial is applied to detect theinitiation and the progression phase change, thereby circumventing thecontamination risk imposed by intra-vial thermometric sensing. Althoughthe thermometric data collected by external vial temperaturemeasurements is subject to variance near the completion of the phasechange due to stochastic movements of the solid sample remnant, locatingthe thermometric sensor to the lower portion of the vial or to the undersurface of the vial would avoid the temperature fluctuations imposed byrandom motion of the solid phase remnant as the solid phase, being lessdense than the liquid phase, will float within the vial thereby beingexcluded from the lower regions of the sample vial. Therefore in someembodiments, the instant invention comprises an external vial surfacethermometric sensor that is located at the mid to upper exterior surfaceof the sample vial while in other embodiments the thermometric sensor islocated at the mid to lower surface, including the undersurface of thesample vial exterior. In some embodiments, external vial surfacethermometry is use to determine the start of the sample phase changewhile in other embodiments, external vial surface thermometry is use todetermine both the start and termination of the phase change.

Now referring to FIG. 14, a time-temperature trace collected from athermocouple in contact with the sample vial exterior at a levelopposite to the approximate mid-sample level of the internal 1 ml sampleduring the thawing cycle process, as described in FIG. 13, is shown. Inthe external temperature trace, as the thermally isolated thermocouple(part 130 in FIG. 1) contacts the −77° C. vial exterior, the temperatureof the sensor declines rapidly until the sensor thermally equilibrateswith the vial exterior at the temperature minimum occurring at the 11second time point. As the temperature of the vial increases, theexternal temperature trace shows a deviation at approximately 60 secondswhere the phase change is known to begin. The trace rises in a shallowslope until approximately 160 seconds where the completion of the phasechange is known to be coincident.

The time-temperature traces collected using an intra-vial temperaturesensor or an exterior surface temperature detector may be divided intothree regions. The first region coincides with the time interval inwhich the contents of the vial are in the solid phase, the secondcoincides with the time interval where the vial contents are mixed solidand liquid phase and the third time interval coincides with the regionwhere the vial contents are liquid phase exclusively. During the firstand third regions, where the vial contents are one of two homogenousphases, the combined mass of the vial and the vial contents behave as alumped capacity system, and the temperature transition behavior may bedescribed by a linear time invariant equation:

$\begin{matrix}{{{T(t)} = {T_{h} + {\left( {T_{c} - T_{h}} \right)e^{\frac{- {({t - t_{pc}})}}{\tau_{v}}}}}},} & {{Equation}\mspace{14mu} 1}\end{matrix}$

wherein T(t), the temperature of the system at time t, may be determinedby the above function wherein T_(h) is the bath temperature, T_(c) isthe starting temperature of the vial, t_(p), is the time offset(required to mathematically match the calculated values to the actualdata plot), and τ_(v) is the effective thermal time constant of the vileand contents. The formula describes the warming of a mass subject to afixed temperature at its outer boundary. An example of fitting thisequation output to the external vial temperature data presented in FIG.14 is shown in FIG. 15.

The warming of the solid phase content of the vial between the pointwhere the sensor reading reaches a minimum at approximately 11 secondsafter the insertion of the vial into the warming block until a time ofapproximately 60 seconds can be closely approximated using the Equation1 above where the value of T_(h) is the warm block temperature (39° C.),T_(c) is the temperature selected at the beginning of the solid phasewarming curve at a time after the vial surface sensor and the vial havereached thermal equilibrium, and at the time point where the value ofthe effective thermal time constant reaches a minimum value (23.8° C.),at 31 seconds past the time of insertion of the vial into the warmblock. The value of the time constant τ_(v) may be calculated from thevalue of T_(h) and the values of T (t) by the following derivation:

$\begin{matrix}{{{T(t)} - T_{h}} = {\left( {T_{c} - T_{h}} \right)e^{\frac{- {({t - t_{pc}})}}{\tau_{v}}}}} & \left. {{Equation}\mspace{14mu} 2} \right) \\{\frac{{dT}(t)}{dt} = {{- \frac{\left( {T_{c} - T_{h}} \right)}{\tau_{v}}}e^{\frac{- {({t - t_{pc}})}}{\tau_{v}}}}} & \left. {{Equation}\mspace{14mu} 3} \right)\end{matrix}$

Therefore,

$\begin{matrix}{\frac{{T(t)} - T_{h}}{\frac{{dT}(t)}{dt}} = {- \tau_{v}}} & \left. {{Equation}\mspace{14mu} 4} \right)\end{matrix}$

By applying a least regression slope analysis of the temperature dataover the time data from a cluster of approximately 5 or 6 time pointsfrom the time-temperature data received from a temperature sensor incontact with the exterior of the vial, the denominator of equation 4 maybe obtained. Likewise, by averaging the vial exterior temperature valuesfrom the same data cluster and subtracting the T_(h) value, thenumerator of equation 4 may be obtained. The τ_(v) value may then beobtained by taking the negative value of the division result. The τ_(v)value obtained by this treatment of the data will only relate to thelinear time-invariant equation that describes the lumped capacity systemof the vial and the solid sample during the portion of the curve priorto the commencement of the phase change, and therefore a deviation fromthe a constant τ_(v) value in excess of a pre-set limit may be used toidentify the beginning of the phase change.

Now referring to FIG. 15A, a time graph of the τ_(v) values for theexample data set shown in FIG. 14 is presented (dark trace). In thevalue plot it can be seen that in the range of 31 to 50 seconds into thewarming process, the τ_(v) values hold a minimum value of approximately55° C. In addition, the time graph is shown (light grey line) of theτ_(v) values obtained by an identical treatment of the output of thelinear time invariant (LTI) equation in which the input values of theconstants were T_(h)=30° C., T_(c)=23.8° C., τ_(v)=55 seconds (asdetermined for the experimental data in FIG. 15), and the t_(pc) timeoffset value is 32 seconds, as determined by a regression analysis fitof the LTI equation to the data set of FIG. 14 in the region of 31 to 50seconds. As may be expected, the τ_(v) value for the LTI equation doesnot change over time. Comparing the two sets of τ_(v) values in FIG.15A, it may be seen that a prediction of the beginning of the phasechange in the contents of the test vial at approximately 50 seconds maybe determined by the deviation of the τ_(v) values for the experimentaldata from the theoretical τ_(v) value. Therefore in some embodiments, adata processing algorithm is embedded into the software of the instantinvention to determine the beginning of the phase change of the contentsof a vial. By adding a time offset value to the beginning of the phasechange time value based upon the following equation, the time of thephase change completion may be estimated. Using equation 5 below, theduration of the thaw (T_(thaw)) may be calculated where ΔH_(f) is thespecific heat of fusion of the sample, m_(soln), is the mass of thesample, R_(v) is the absolute thermal resistance of the sample vialwall, T_(vial) is the temperature of the vial exterior wall, and T_(m)is the melting temperature of the sample.

$\begin{matrix}{T_{thaw} = \frac{\Delta \; {H_{f} \cdot m_{{so}\; \ln} \cdot R_{v}}}{T_{vial} - T_{m}}} & \left. {{Equation}\mspace{14mu} 5} \right)\end{matrix}$

As a typical biological sample stored in a cryogenic sample vial is anaqueous solution, the melting temperature is not a single value as wouldbe the case for a homogenous material, but rather a temperature range.Nevertheless, refinement of equation 5 by experimental determination ofa value of T_(m), for a specific vial, that will allow a fit with theactual T_(thaw) value will provide a more accurate means of predictingthe T_(thaw) value for different sample masses. However, as was shown inFIGS. 12 and 13, the rate of thermal energy influx in a thawing samplecontained within a sample vial is largely independent of the samplevolume and therefore of the sample mass. Therefore, in some embodimentsof the instant invention the termination of the phase change isdetermined by a software algorithm that combines the time valuecalculated for the start of the phase change as described above with anexperimentally derived phase change duration value for a given samplevial to determine the phase change completion time. In other embodimentsthe reduction of the determined phase change completion time value by aconstant will be used to terminate the thawing process while some solidphase is still remaining in the vial.

Now referring to FIG. 15B, an alternative data treatment by which thebeginning of the phase change may be determined is shown. In FIG. 15A,part A, the plot of the experimental vial exterior temperature describedin FIG. 14 is indicated along with the warming block temperature tracefor the same experiment. A plot of a LTI equation output values thatoverlays the solid phase portion of the warming sequence is shownimposed on the experimental data. The variable values for the LTIequation as described in equation 1 above were derived from the warmingblock temperature (T_(h)=3 9° C.), the minimum τ_(v) value following thevial surface temperature sensor equilibrium, as determined by equations2-4 above, and the temperature of the vial surface at the time where theminimum τ_(v) value was first observed (T_(c)=23.8° C.). The t_(pc)value was determined by iterative refinement such that a minimum valuewas identified for the difference between the LTI equation output and alinear regression analysis of vial surface temperature data pointsfollowing the time of detection of the minimum τ_(v) value.

Now referring to FIG. 15B, part B, a graph of the difference between theexperimental data and the output of the LTI equation is shown withrespect to time. The vertical arrow located at approximately 60 secondsindicates the point where the experimental data and the calculated LTIequation data diverge by a selected value of 0.2° C. In some embodimentsof the instant invention, the embodiment of the above divergence gate inthe algorithmic software will determine the beginning of the phasechange.

Now referring to FIG. 16, without limitation, the dimensions of threesample vial types that may be used the instant invention are shown. Insome embodiments of the invention, the warming blocks (2008 and 2010 insubsequent FIG. 20) may be adjusted in the dimensions of the vialreceiver well (2033 in subsequent FIG. 20) to accommodate the differencein the sample vial dimensions shown, thereby allowing the adaptation ofa common design of the instant invention to be applied to multiplesample vial types. The sample container may be any cryogenic vial. Forexample, the sample container may be a standard 5.0 mL vial, 4.0 mLvial, 2.0 mL vial, 1.2 mL vial, 500 μL vial, etc. The vials may beconstructed out of polypropylene or other materials, for example, andmay be round bottomed or self-standing. The sample holder may be inheat-transferring contact with the side-walls of a received vial.Optionally, the sample holder may be configured to limit the heattransfer from the top or bottom of a sample container. While the samplecontainer is generally described as a vial, it should be understood thatsamples in other containers may be thawed using methods and systemsdescribed herein. For example, the container may be a bag or othervessel as desired.

In other embodiments, the warming blocks may comprise a single vialreceiver well dimension that may be fit to accommodate multiple vialtypes and dimensions by comprising an adaptor part.

Now referring to FIG. 17, a the exterior of a representative embodimentof the invention is shown. The embodiment is presented as a functionalexample of the invention only and is not intended to limit alternativeembodiments of the invention. The figure identifies the cover for theinternal mechanism that comprises an external upper shell 1710 and aclear or translucent base 1720. In this embodiment, the uses inserts asample vial 1740 into the available top opening 1730 to begin thewarming sequence. When the vial insertion achieves a specific depth, theinternal mechanism triggers the rapid closure of an open segmented warmblock that will contact and hold secure the sample vial during thewarming process.

Now referring to FIG. 18, two views of an embodiment of the internalmechanism are shown. In this embodiment, the main frame 1810 isconnected to a two part warming block 1820, the two parts of which areidentical and joined by a 180-degree rotation of one part relative toits mating part. The two block halves are joined by a hinge pin 1825such that the two warming block parts may rotate and separate to a10-degree included angle between the vertical plane median faces of theparts. The two block halves are articulated about the hinge axis 1825 bya sliding spreader frame 1815 which engages pin shafts 1817, embedded inthe warming block parts 1820 on both sides of each part, through anglesslots 1819 such that when the sliding spreader frame 1815 ascends anddescends relative to the main frame 1810, the slots 1819 open and closethe warming block halves 1820. The sliding spreader frame 1815 isarticulated by two solenoid actuators 1850, one configured to elevatethe spreader frame 1815 when activated and one configured to de-elevatethe spreader frame relative to the main frame 1810. The two warmingblock halves 1820 comprise a central vial receiving well 1865 which isdivided in a vertical plane that is coincident with the vertical planeseparating the two warming block halves. The central vial well extendsthrough the entirety of the warming blocks and comprises a 2 mm-thicklining of thermally conductive foam 1860 that covers the entire innersurface of the central vial well to a depth of 1.1 inches below the topsurface of the warming block. A thermistor temperature sensor 1835 isembedded in both of the warming block parts and the temperature datasignals are conducted to the microprocessor board 1805 via wireconnections (not shown). One or both of the warming blocks comprise anadditional temperature sensor 1830 that is thermally isolated from thewarming block 1820 and rests in contact with the exterior surface of thesample vial 1870 when the sample vial is inserted into the central vialwell and the warming block jaws 1820 are closed. The data signal fromthe sensor 1830 is conducted to the microprocessor board 1805 byconnector wires (not shown).

Now referring to FIG. 19, the overall dimensions of the embodiment shownin FIG. 18 are shown. The exterior shell shown in part A has a height of5 inches and a major diameter of approximately 4 inches. The top openingaccess for the vial receiver well has a diameter of approximately 0.6inches. The in inner mechanism shown in part B has an overall height ofapproximately 4.9 inches, a major diameter of approximately 4 inches,and a vial receiving well diameter of approximately 0.5 inches.

Now referring to FIG. 20, an exploded view of the embodiment shown inFIGS. 18 and 19 is shown. In this diagram, the main frame 2002 supportsthe remaining parts of the internal mechanism and is joined to the basepart 2003 by fasteners (not shown) that extend through themicroprocessor circuit board 2004. The two warming block halves 2008 and2010 are joined to the main frame 2002 by the hinge pin 2012. Thewarming block halves rotate on the hinge pin 2012 and are therebyrestricted to a range of motion from a closed position in which themedian plane faces are parallel to an open position where the medianplane faces are separated with an included angle of 10 degrees. The armextensions 2009 of the warming block halves comprise cylindrical holesthat may optionally receive cylindrical magnets (not shown) that wheninstalled will mate with magnets installed on the opposite warming blockpart in a manner that will provide a holding force hold the warmingblock jaws in either an open or closed position in the absence ofarticulation or holding forces applied by other components in theembodiment. The two warming block jaws 2008 and 2010 are articulated bya sliding spreader frame 2006 that engages the warming blocks throughangled slot features 2017 that engage pin bearings 2016 that areembedded in the warming block recesses 2018. The slots 2017 in thesliding spreader frame are angled such that when the sliding spreaderframe is raised relative to the main frame the warming blocks rotate onthe hinge pin 2012 and open to an angle of 10 degrees. When the slidingspreader frame is lowered relative to the main frame, the warming blocksrotate to a closed orientation where the inner vertical face areparallel. The position of the sliding spreader frame in relation to themain frame is monitored by an optical sensor 2048 that detects a lightsignal from a light source 2046, both of which are mounted to the mainframe. When the sliding spreader frame is raised, the light signal fromthe source 2046 has an unobstructed path through the slot 2049 in thesliding spreader frame and through the open warming block parts providedthere is no other obstruction in the central vial receiver such as asample vial. When the sliding spreader frame is lowered in relation tothe main frame, the light from source 2046 is blocked by the slidingspreader frame. The light source 2046 and the light detector 2048receive power from the microprocessor circuit board 2004 through powerwires (not shown) and the microprocessor receives a digital signal fromthe optical sensor 2048 through wire conduits (not shown). The slidingspreader frame 2006 is articulated by solenoid activators, one thatelevates the spreader frame when activated 2052, and one thatde-elevates the spreader frame when activated 2050. The solenoidactuators are joined to the sliding spreader frame through an L-bracket2038 that is fastened to the spreader frame and to the main framethrough holes 2043 and fastened by a hex nut 2044. Two thermallyconductive foam pads 2030 and 2032 line the two halves of the centralvial receiving well 2033 in the warming blocks 2008 and 2010. One ormore of the foam pads 2030 and 2032 comprise a passage 2035 throughwhich a temperature sensor 2042 embedded in the warming block(s) maypass through to contact the exterior surface of a sample vial containedbetween the foam pads 2030 and 3032 within the vial receiving well ofthe blocks. Each of the warming blocks 2008 and 2010 comprise one ormore heater elements 2034 that is received into a cavity in theunderside of the warming block parts (not visible). The temperature ofthe warming blocks 2008 and 2010 is sensed by one or more temperaturesensors 2040 that are embedded in the warming block in a receiver cavity2041 and secured by a perpendicular set screw (not shown). A centralpedestal 2024 is positioned in a coaxial orientation to the central vialreceiving well and is mounted on a sliding support 2026 that comprises aslot through which the hinge pin 2012 passes thereby capturing andlaterally constraining the sliding support. The sliding support isfurther restrained and supported by two bushing bearings 2028 throughwhich the hinge pin passes and that are positioned on either side of thesliding support flat surfaces. The sliding support is furtherconstrained to a vertical linear motion by joining with the slidingshaft of a motion damper 2054 that is mounted to the underside of themain frame 2002. The sliding support 2026 further comprises a notch andhole 2029 which, depending on the elevation of the part, allows orblocks an light signal of an optical sensor 2036 that is mounted on theL-bracket 2038. Depending upon the relative positions of the slidingpedestal support 2026 and the sliding spreader frame 2006, a high andlow digital signal from the optical sensor 2036 may indicate one of fourpositions states when combined with the vertical position signal fromthe sliding spreader frame position sensor 2048: 1) sliding support 2026raised; sliding spreader frame 2006 raised, 2) sliding support 2026lowered; sliding spreader frame 2006 raised, 3) sliding support 2026lowered; sliding spreader frame 2006 lowered, and 4) sliding support2026 raised; sliding spreader frame 2006 lowered (a fault condition asdescribed in subsequent figures). The damper 2054 in some embodimentscomprises a spring (not shown) that is configured such that when thepedestal 2024, the sliding support 2026, and the damper shaft arelowered, the spring is compressed and provides a force that can restorethe parts to the lifted position. In some embodiments the lifting motionresulting from the spring force opposed only by the friction force ofthe conductive foam 2030 and 2032 contact with the sample vial exterior,while in other embodiments, the lifting spring force is controlled byactively regulated mechanical restrictors, such as, but not limited to,solenoid latches. In alternative embodiments, the lifting force isprovided partially or exclusively by actively controlled actuators suchbut not limited to solenoids and motors. Some embodiments comprise auser interface including, but not limited to, LED lights and lightarrays, LCD screens, keypads, button switches, sliding switches, touchscreens, knobs, slide switches, capacitive switches, and wirelesslinkage to remote control interfaces. In the embodiment shown in FIG.20, a radial array of LED lights 2080 is fixed to the main frame thatwill be visible through the outer shell (not shown) through translucentshell material. The LED array illumination may be controlled by themicroprocessor board 2004 through a ribbon wire connector (not shown)and may indicate the thawing status of the sample vial, states ofreadiness, and error codes. While illustrated with the lights positionedon a top surface of the device, it should be understood that otherembodiments may include an LED array on a side of the device. In someembodiments, the microprocessor board comprises data ports by which themicroprocessor may transmit stored data or may receive a data streamfrom an external source, for example for the purpose of installingsoftware updates.

Now referring to FIGS. 21-23, an example of the operation sequence ofthe embodiment shown in FIGS. 19 and 20 is shown. In FIGS. 21-23, theembodiment is displayed with the forward warming block removed so thatthe internal mechanism and position of the parts may be illustrated.Following previous temperature equilibration to −77° C. by placing thesample vial 2001 in a temperature equilibration apparatus such as thosedescribed in the embodiments shown in FIGS. 2-5 for a period of ten ormore minutes, the thawing cycle is initiated by insertion of a samplevial 2001 into the warming block vial receiving cavity as shown in FIG.21, part A. In this state, the warming block has been previouslytemperature-equilibrated to the appropriate warming temperature, thesliding support frame 2006 is raised and the warming blocks 2008 and2010 (not shown) are open. In thaw cycle step 1, shown in FIG. 21, partB, the sample vial is manually depressed, lowering the pedestal 2024 andsliding support 2026 until the sliding support optical trigger 2029passes the optical sensor 2036. Now referring to FIG. 22, part A.Sensing that the sample vial has descended to the appropriate depth, themicroprocessor board activates the solenoid (2050 in FIG. 20) to lowerthe sliding spreader frame 2006, thereby closing the warming blocks onthe sample vial 2001, initiating the process of thermal energy transferto the vial and contents.

Now referring to FIG. 22, part B, the thawing process having beencompleted, the microprocessor activates the lifting solenoid (2052 inFIG. 20) to raise the sliding spreader frame 2006, thereby opening thewarming blocks 2008 and 2010 (not shown) releasing the restrainingfriction between the conductive foam liner, 2030 (not shown) and 2032,and the vial. The opening of the warming blocks disrupts the thermalconduction pathway from the conductive foam thereby preventing orsignificantly delaying an undesired temperature rise in the vialcontents.

Now referring to FIG. 23, part A, the friction restraint upon the vialnow disrupted, the sliding support 2026 and the vial support pedestal2024 now raise and present the sample to the operator and as the slidingsupport optical trigger 2029 passes the optical sensor 2036, themicroprocessor receives a signal that the vial has been presented. Nowreferring to FIG. 23, part B, when the vial is removed from the warmingblock vial receiver, the light pathway between the optical light source2046 and the optical sensor 2048 becomes unobstructed and themicroprocessor receives a signal that indicates that the vial has beenremoved, thereby preventing algorithmic activation of alert and alarmsignals.

Now referring to FIG. 24, a graphic plot time-temperature plot of athawing experiment series is shown to demonstrate the effectiveness ofopening the warming blocks upon the termination of the thawing processin interrupting the thermal energy flow into the sample. In thisexperimental series, repeated cycles of vial freezing in liquidnitrogen, equilibration in the apparatus described in FIGS. 2 and 3, andinsertion into a 45 degree warming block of the design described inFIGS. 7 and 8, and the internal temperature of a 1 ml sample payloadmonitored by the insertion of a thermocouple sensor held in a positionnear to the internal wall of the vial to a position at half the heightof the sample. In one test cycle, upon termination of the thaw asdetermined by time measurement, the warming blocks were left closed andthe vial allowed to remain in the warming block for a total interval ofapproximately 6 minutes. In this experiment, the temperature of thesample continues to rise toward the block temperature. In a second cycleseries, the sample vials upon completion of the thaw, the warming blockwas opened and the vials were removed from the warming block and held inopen air for a total duration of approximately 6 minutes. In this dataset, the temperature of the sample increased very little over thethree-minute interval following the block opening. In a third cycleseries, the previous experiment was repeated with the exception that thevial was allowed to remain in the warming block following the opening ofthe warming block. In this series, the temperature of the vial contentsincreased at a slightly higher rate that when the vial was removed fromthe block upon the termination of the thaw, however the increase intemperature was significantly less than that observed when the warmingblock was left closed upon termination of the thaw. The experiment setstrongly supports the benefit of interrupting the thermal conductionpathway between the warming block and the sample vial to terminate thethermal energy influx into the sample upon the completion of the thawprocess. In some embodiments of the instant invention, the warmingprocess following insertion of a sample vial into the warming blockreceiving well is terminated by the introduction of air space betweenthe solid material of the warming block and the exterior surface of thesample vial. In some embodiments, the system may be configured tomaintain the sample at a desired temperature at the end of the thawing.

In some embodiments, multiple algorithms may be provided for determininga thaw end time. Optionally, each of the multiple algorithms may beconcurrently run to provide separate estimates for the thaw end time.The system may be configured to end the thawing based on the algorithmwhich first provides an estimated thaw end time. Optionally, the systemmay be configured to allow each of the algorithms to complete theirestimations and may utilize the shortest thawing interval calculated. Infurther embodiments, a system may be configured to average the estimatedthawing intervals and utilize the averaged thawing interval to determinethe thaw end time.

Different arrangements of the components depicted in the drawings ordescribed above, as well as components and steps not shown or describedare possible. Similarly, some features and sub-combinations are usefuland may be employed without reference to other features andsub-combinations. Embodiments of the invention have been described forillustrative and not restrictive purposes, and alternative embodimentswill become apparent to readers of this patent. Accordingly, the presentinvention is not limited to the embodiments described above or depictedin the drawings, and various embodiments and modifications may be madewithout departing from the scope of the claims below.

What is claimed is:
 1. A method of thawing a sample within a vessel, themethod comprising: receiving a temperature data feed from one or moretemperature sensors reporting an exterior surface temperature of thevessel at a location along the exterior surface of the vessel that isbelow a top level of a sample within the vessel; calculating a thaw endtime based on the temperature data feed received from the one or moretemperature sensors; and outputting a signal to interrupt thawing of thesample contained in the vessel at the calculated thaw end time, whereinthe sample at the thaw end time has solid phase remaining in an aqueoussolution in the vessel.
 2. The method of claim 1, wherein calculatingthe thaw end time is further based on a calculated time value for thestart of a phase change from solid to liquid, and based on a phasechange duration for the vessel.
 3. The method of claim 2, furtherincluding: determining a first thawing stage interval by recording aheater contact time at which an operating heater contacts the samplecontainer, and by subtracting the heater contact time from thecalculated time value for the start of the phase change from solid toliquid; adding the phase change duration for the vessel to the firstthawing stage interval to determine a calculated total thawing duration;and interrupting the thawing process when a measured time durationstarting from the heater contact time exceeds the calculated totalthawing duration.
 4. The method of claim 3 wherein as a result ofoutputting a signal to interrupt thawing of the sample contained in thevessel a heater in contact with the sample container separates from thesample container.
 5. The method of claim 3 wherein parallel algorithmicprocesses may modify the value calculated for either or both of thefirst thawing stage interval and the phase change interval.
 6. Themethod of claim 2, further comprising determining the calculated timevalue for the start of the phase change from solid to liquid byidentifying a significant change in a first derivative of a warmingcurve defined by the recorded temperature measurements.
 7. The method ofclaim 1, wherein the temperature data feed received from the sensor isused to calculate a timing event associated with a change in state fromsolid to liquid of the sample contained in the vessel, and calculatingthe thaw end time is based on the timing event.
 8. The method of claim1, wherein the temperature data feed received from the sensor is used tocalculate the rate of temperature increase of the vessel, and the thawend time is based on the rate of temperature increase.
 9. The method ofclaim 1, wherein the step of calculating the thaw end time of the samplecomprises adding an average thaw time to a calculated time value. 10.The method of claim 9, wherein the step of calculating the thaw end timecomprises accessing a library of average thaw times.
 11. The method ofclaim 10, wherein the library comprises average thaw times for aplurality of different container types.
 12. The method of claim 10,further comprising the step of identifying the sample container type andaccessing a library of average thaw times for the sample container type.13. The method of claim 1, further comprising providing a signal alertto a user based on the thaw end time.
 14. The method of claim 13,wherein the signal alert is an audio and/or a visual alert.
 15. Themethod of claim 1, further comprising adjusting the thaw end time basedon a sample container material.
 16. The method of claim 15, furthercomprising adjusting the thaw end time based on a presence of a samplecontainer label.
 17. A method for thawing a cryogenic sample in a samplecontainer, the method comprising: heating a sample container with aheater; determining a thaw start time for the cryogenic sample;determining an estimated thaw end time of the cryogenic sample based onthe determined thaw start time of the cryogenic sample, the cryogenicsample at the estimated thaw end time having solid phase remaining in anaqueous solution in the sample container; and stopping the heating ofthe sample container with the heater after the estimated thaw end time.18. The method of claim 17, further comprising: measuring a temperatureof an outer surface of the sample container; and recording thetemperature measurements.